Turkish Journal of Biology

Turk J Biol
(2015) 39: 461-468
TBTAK
doi:10.3906/biy-1408-47

http://journals.tubitak.gov.tr/biology/

Research Article

Aspects of mycorrhizal colonization in adaptation of sweet marjoram

(Origanum majorana L.) grown on industrially polluted soil
1,

Marieta HRISTOZKOVA *, Maria GENEVA , Ira STANCHEVA , Madlen BOYCHINOVA , Efrosina DJONOVA
Department of Plant Mineral Nutrition and Water Relation, Institute of Plant Physiology and Genetics, Bulgarian Academy of
Sciences, Sofia, Bulgaria
2
Department of Soil Microbiology, N. Pushkarov Institute of Soil Science, Agrotechnologies and Plant Protection, Sofia, Bulgaria
1

Received: 22.08.2014

Accepted/Published Online: 14.01.2015

Printed: 15.06.2015

Abstract: The current study reveals the effects of mycorrhization on heavy metal uptake and accumulation, antioxidant potential, and
essential oil composition of Origanum majorana L., grown on soil polluted with industrial Cd and Pb. Two strains of Claroideoglomus
claroideum (EEZ 54 and EEZ 55) were isolated from soil that is naturally rich in metals. EEZ 35 (Funneliformis mosseae) was isolated
from a place with industrial contamination. The percentage of mycorrhizal colonization with the EEZ 54 strain was higher than those
of the other strains; there was no significant difference between EEZ 55 and EEZ 35. The highest value of the total identified essential
oils was observed in plants inoculated with EEZ 35, where Pb accumulated in the roots. Mycorrhizal colonization led to a change in the
content of the main compounds of marjoram essential oil. The EEZ 54 and EEZ 55 strains, isolated from areas with naturally high levels
of metals, significantly reduced Pb accumulation in marjoram shoots and roots as compared with nonmycorrhizal plants. Antioxidant
activity in marjoram aerial parts increased as a result of inoculation with EEZ 54 and EEZ 35 arbuscular mycorrhizal fungi strains due
to the elevated levels of phenolic compounds.
Key words: Origanum majorana L., essential oil composition, food quality, antioxidant capacity, mycorrhizal colonization, industrially
polluted soil

1. Introduction
Contamination of soils with heavy metals is a major
environmental problem in the world and limits usable
agricultural land. High concentrations of heavy metals
such as cadmium, lead, copper, and zinc due to industrial
activities cause environmental pollution because they have
a strong persistence and exert detrimental effects on plant
functions. Monitoring for toxic heavy metals in medicinal
plants has become part of the quality control in the
pharmaceutical industry as consumers demand products
that are free from potentially harmful constituents
(Chizzola et al., 2003).
The role of mycorrhiza in metal stress attenuation is
well recognized; improved nutritional status and reduced
metal uptake are among the most common benets
for host plants. The alleviation of metal toxicity can be
attributed to the impact of arbuscular mycorrhizal fungi
(AMF) on metal distribution at the soilfungusplant
interface (Meier et al., 2012). AMF may stabilize metals
in the soil, reduce their uptake, and thus decrease the
risk of toxicity to plants growing in polluted substrates
* Correspondence: mhristozkova@abv.bg

(Gonzlez-Chvez et al., 2009). Mycorrhizal fungi can

affect the transformation of trace metals in the soil in
several ways, including: 1) altering the pH of the soil
(i.e. acidification); 2) immobilization (by adsorption,
chelation, or absorption of free metallic species in the soil
solution); 3) modification of the root exudation; 4) hyphal
sequestration; and 5) chemical precipitation of metal in the
soil (Joner et al., 2000; Gonzlez-Chvez et al., 2002; Bai et
al., 2008; Giasson et al., 2008; Gadd, 2010; Upadhyaya et
al., 2010).
There is a lot of research on the phytoextraction
potential of medicinal and aromatic plants grown as
alternatives to edible crops in heavy metal polluted
agricultural soils (Pavlovi et al., 2006; Zheljazkov et al.,
2006, 2008; Doumett et al., 2008; Kovaik et al., 2009;
Stancheva et al., 2010, 2014). However, the role of AMF in
reducing heavy metal stress in medicinal plant tissues has
not been extensively studied.
Among the medicinal and culinary plants, sweet
marjoram (Origanum majorana L., Lamiaceae) is of great
economic and industrial importance (Werker et al., 1993).

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HRISTOZKOVA et al. / Turk J Biol

Marjorams fresh and dried aromatic leaves and flowers

are used to flavor many foods. Marjoram oil possesses
antimicrobial, antiinflammatory, and antispasmolytic
properties (Ezzeddine et al., 2001; Jun et al., 2001). O.
majorana is distinguished by its strong antioxidant
potential, mainly because of its high concentrations of
phenolic acids and flavonoids, which are useful in food
supplements and preservation (Vagi et al., 2005). The
effects of root colonization by AMF (Glomus mosseae) on
the qualitative and quantitative pattern of essential oils
were determined in three oregano genotypes (Khaosaad
et al., 2006). It was established that two oregano genotypes
showed a clear increase in the essential oil content through
mycorrhization. In addition, studies on Ocimum basilicum
(Copetta et al., 2006) and Mentha arvensis (Freitas et al.,
2004) showed that arbuscular mycorrhiza root colonization
increases the essential oil content in both species.
The aim of the present study was to determine whether
three strains of AMF, isolated from contaminated areas,
can reduce metal accumulation in plant organs, and to
evaluate how accumulated metals affect the antioxidant
potential and essential oil composition of O. majorana L.
grown on industrially polluted (with Cd and Pb) soil.
2. Materials and methods
2.1. Biological materials and growth conditions
O. majorana L. plants were grown from seeds in glasshouse
conditions under natural sunlight for 90 days, from April
to July. The glasshouse temperature was between 15 C and
30 C, and the relative humidity ranged from 40% to 65%.
The plants were harvested in the middle of July, during the
flowering growth stage when the quality and quantity of
essential oils is the highest (Sellami et al., 2009). The plants
were grown in 1.2 kg plastic pots (3 plants per pot) on the
soil/sand substrate in a ratio of 3:1, in four replicates for
each variant. All pots were adjusted daily to 60% water
holding capacity. The polluted soil was taken from a field
near the waste depository of a ferrous metallurgical plant
and had the following agrochemical characteristics: pH
7.94, 10 mg kg1 soil total mobile nitrogen (N NO3 +
N NH4+), 36.8 mg kg1 soil P2O5, 308 mg kg1 soil K2O.
The following concentrations of the studied HMs (mg
kg1 DW) were measured: Cd4.8, Pb142, and Zn207.
The Bulgarian permissible limit concentrations (PLC) at
pH 7.94 are: Cd < 3.0, Pb < 120, and Zn < 400 mg kg1
DW. Thus, the studied soil is polluted with Cd and Pb.
Three cultures of AMF were used: 1) Strain EEZ 35:
Claroideoglomus claroideum, (N. C. Schenk & G. S.
Sm.) Walker & Schler (syn. Glomus claroideum N. C.
Schenk & G. S. Sm.) isolated the rhizosphere of Zea mays
in Braunchweig (Germany), in a parcel contaminated
by the repeated addition of sludge; 2) Strain EEZ 54: a
different isolate of Claroideoglomus claroideum from Rio

462

Tinto (Huelva, Spain) from the rhizosphere of Lavandula

stoechas; 3) Strain EEZ 55: Funneliformis mosseae, (Nicol.
& Gerd.) Walker & Schler (syn. Glomus mosseae (Nicol.
& Gerd.) Gerd & Trappe), (Schler and Walker, 2010)
also isolated it from Rio Tinto (this soil naturally has some
quantity of metals). Inoculation with AMF was done at the
seeding by the layering method (Jackson et al., 1972).
Four variants were tested: 1 - control nonmycorrhizal
plants (NM); 2 - EEZ 35; 3 - EEZ 54; and 4 - EEZ 55.
2.2. Determination of root colonization
The percentage of mycorrhiza infection of the roots was
determined microscopically (Giovanetti and Mosse, 1980).
To visualize the AMF colonization, roots were cleared by
boiling for 4 min in 10% KON, rinsed three times with
tap water, and acidified with 0.1% HCl. Roots were stained
boiling in 0.05% Trypan blue for 10 min. Following
staining, the roots were rinsed with tap water, mounted on
slides, and observed directly in lactic acid.
2.3. Heavy metal analysis
The heavy metal content was analyzed in soil samples
before sowing and immediately after plant harvest. The
plant samples were digested in a solution containing
3:1 (v/v) HNO3:HClO4. The samples were heated at
200 C to evaporate to dryness. The residue was taken
up in 25 mL of 1 N HCl (Doumett et al., 2008). Heavy
metal concentrations were determined using an atomic
absorption spectrophotometer (AAS, PerkinElmer 2100,
USA).
2.4. Antioxidant capacity assays
2.4.1. Total antioxidant capacity
Free radical scavenging activity was measured from the
bleaching of the purple methanol solution of free stable
radical (diphenylpycril-hydrazyl, DPPH), according
to Tepe et al. (2006). DPPH is a stable radical with a
maximum absorption at 517 nm that can readily undergo
reduction by an antioxidant. The percent inhibition of
the DPPH radical (I%) was calculated by the following
equation:
I% = (Ablank Asample/Ablank) 100,
where Ablank is the absorbance of the control reaction
(containing all reagents except the test compound), and
Asample is the absorbance of the test compound, i.e. puncture
vine extracts.
2.4.2. Ferric reducing power (FRAP assay)
The FRAP reagent was freshly prepared by mixing acetate
buffer (300 mM, pH of 3.6), TPTZ solution (10 mM TPTZ
in 40 mM HCl), and FeCl3-6H2O (20 mM) in a ratio of
10:1:1 (Benzie et al., 1996). To perform the assay, 900 L of
FRAP reagent, 90 L of distilled water, and 30 L of plant
extract were mixed and incubated at 37 C for 15 min. The
absorbance was measured at 595 nm using FRAP working
solution as a blank. The antioxidant potential of samples

HRISTOZKOVA et al. / Turk J Biol

2.4.3. Determination of the phenolic compounds and

flavonoids
Dry leaves samples (1 g) were ground and exhaustively
extracted with 96% (v/v) methanol. Concentrations
of
phenolic
compounds
were
determined
spectrophotometrically using FolinCiocalteu reagent
and calculated as caffeic acid equivalents (Pfeffer et
al., 1998). Flavonoids in plant tissues were measured
spectrophotometrically according to Zhishen et al. (1999),
using the standard curve of catechin.
2.5. Essential oil analyses
Plant samples were taken early in the morning by
randomized collection of 5 individuals per treatment.
The air-dried and finely ground leaves of each treatment
were subjected to a microsteam distillation/extraction in
a Likens-Nickerson apparatus with modification for 2 h
(Berkov et al., 2011). The essential oil compounds of the
steam distilled oil were determined by GC and GC/MS
analyses. GC analyses were carried out on an HP 5890 gas
chromatograph (FID), carrier gas nitrogen, linear velocity
25 cm/s, fused silica capillary column HP 1.30 m 0.25
mm, df = 0.25 m. The injector and detector temperatures
were 260 C, and column temperature was programmed
from 50 C to 230 C at a rate of 4 C/min and 15 min at
260 C. The GCMS spectra were recorded on a Hewlett
Packard 6890+MSD 5975 (Hewlett Packard, Palo Alto, CA,
USA) operating in EI mode at 70 eV. A DB-5 MS column
(30 m 0.25 mm 0.25 m) was used. The temperature
program was: 100180 C at 15 C min1, 1 min hold at 180
C, and 180300 C at 5 C min1, and 1 min hold at 300
C. Injector temperature was 280 C. The flow rate of the
carrier gas (helium) was 0.8 mL min1. The split ratio was
1:15. Ions atm/z 287, 286, and 174 were used to collect SIM
chromatograms in SIM mode.
The components were identified by comparison of
their mass spectra and retention indices with appropriate
standards.
2.6. Statistics
Data are expressed as means standard error, where the
number of repetitions varied between 3 and 10, depending
on the type of analysis. Comparison of means was
performed by the Fisher least significant difference (LSD)
test at P 0.05 following ANOVA. A statistical software
package (StatGraphics Plus, version 5.1 for Windows,
USA) was used.

3. Results
The percentage of mycorrhizal colonization in the control
variants may be due to the natural diversity in the soil
of AMF being different from the referred species in the
experiment. It could be suggested that the higher number
of AMF structures (as compared with the control) was a
result of colonization of the applied three strains (Figure
1). The mycorrhizal status is best manifested in the plant
roots inoculated with Claroideoglomus claroideum EEZ
54. The percentage of root mycorrhizal colonization
with strain EEZ 54 was significantly higher than those
of EEZ 55 and EEZ 35; no significant difference between
the colonization percentages of EEZ 55 and EEZ 35 was
observed.
Cd concentration in the soil following plant harvesting
was under 1 mg kg1 and differences among the
experimental variants were not detected. Very low levels
of Cd were measured in the marjoram shoots and roots
both in the control and the AMF infected plants (Table
1). In the shoots of plants inoculated with EEZ 55, Cd
concentration was under 0.5 mg kg1 and therefore not
detectable; Cd concentration in the roots of those plants
was also estimated to be undetectable. Similarly low levels
(slightly above the undetectable ones) were observed
in the roots of the other variants. Pb residues in the soil
were lower as a result of inoculation with EEZ 55. Lead
concentration was higher in the O. majorana roots than
in the shoots except for the EEZ 55 inoculated plants,
where its concentration was equally distributed between
the roots and the shoots (Table 1). Significant reduction
of Pb compared with the controls was observed both in
50
b

COLONIZATION (%)

was determined from a standard curve, plotted using the

FeSO4.7H2O linear regression. The results were corrected
for dilution and expressed as mol of Fe2+ g1 of dried
sample. All assays were determined in triplicate.

40
30

20
10

NM

EEZ35

EEZ54

EEZ55

TREATMENTS

Figure 1. Percentage of colonization of O. majorana roots,

infected by three AMF strains (EEZ 35, EEZ 54, EEZ 55). Values
are means SE, n = 9; letters in common within a graph indicate
no significant differences assessed by Fisher LSD test (P 0.05)
following ANOVA.

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Table 1. Concentrations of Pb and Cd in soil and plant organs following the harvest of O. majorana L. plants.

Variants

Soil

Shoots

Roots

Pb mg kg DW

Cd mg kg DW

Pb mg kg DW

Cd mg kg DW

Pb mg kg 1DW

Cd mg kg1DW

NM

88.5b 4.425

less than 1

7.86c 0.393

less than 0.5

13.3c 0.665

0.75

EEZ 35

90.5b 4.525

less than 1

8.03c 0.402

less than 0.5

18.7d 0.935

0.75

EEZ 54

85.5ab 4.275

less than 1

3.32a 0.166

less than 0.5

10.7b 0.535

0.75

EEZ 55

78.0 3.9

less than 1

5.48 0.274

less than 0.5

4.46 0.223

less than 0.5

LSD

8.037

0.608

1.210

Values are means SE, n = 6; the same letter within a column indicates no significant difference assessed by Fisher LSD test (P 0.05)
following ANOVA.

16

24

bc

c
a

12

16

DPPH

FRAP

100

antioxidant capacity (%)

ab

b
a

75

50

25
2

NM

EEZ35

EEZ54

EEZ55

NM

EEZ35

EEZ54

1
concentration (mg gDW )

flavonoids

32

concentration (mg gDW )

phenols

significant differences were not found. Flavonoid

concentrations showed maximal values as a result of EEZ 54
inoculation. According to the DPPH test, aerial marjoram
parts have very high free radical scavenging activity
(about 90%). No differences between the experimental
variants were found. FRAP highlighted higher antioxidant
capacity in the plants inoculated with EEZ 35 and EEZ 54.
DPPH and FRAP assays are frequently used to measure

ferric reducing power (mol gFW

the shoots and the roots of marjoram inoculated with EEZ

54 and EEZ 55 strains. Plants inoculated with EEZ-35
accumulated Pb at higher levels than the controls.
The antioxidant activity of O. majorana aerial part
was assessed by different in vitro tests. Total phenol
concentrations in the methanol plant extracts showed
significantly lower values only in EEZ 55 plants (Figure
2). Among the other experimental variants, statistically

EEZ55

TREATMENTS

Figure 2. Total phenolic and flavonoid concentrations and antioxidant activity of O. majorana aerial parts.
Values are means SE, n = 9; letters in common within a graph indicate no significant differences assessed by
Fisher LSD test (P 0.05) following ANOVA.

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terpene4-ol, and a- and g- terpinene as compared with

the other variants (and NM). The EEZ 54 strain increased
g-terpinene, and EEZ 55 increased the percentages of
sabinene, trans-sabinene hydrate acetate, and linalyl
acetate. The compounds terpene-4-ol, cis-sabinene
hydrate, and g-terpinene were the most abundant in all
experimental variants. In NM plants these constituents
were followed by -terpinene and -terpineol. In variants
treated with EEZ 35 and EEZ 55, they were followed by
sabinene and linalyl acetate, and in plants inoculated with
EEZ 54 they were followed by -terpinene and sabinene.
The highest value of the total identified essential oils was
observed in plants inoculated with EEZ 35.

the total antioxidant capacity of plant extracts (Figure 2).

Correlation coefficient tests between antioxidant activity
(DPPH and FRAP) and total phenols and flavonoids in
above-ground plant parts of O. majorana were performed.
Higher correlation coefficients (P 0.05) in the range of
0.7390.953 were obtained for FRAP compared with the
DPPH test, which ranged from 0.582 to 0.846 (Table 2).
Our results indicate that O. majorana antioxidant potential
is mainly defined by its high concentration of phenolic
compounds and flavonoids.
The full composition of essential oils in sweet marjoram
was identified with the retention indexes of 70 compounds
(data not shown). The major components in O. majorana
essential oils (>5% for the variants) were determined as
terpene4-ol (23%), cis-sabinene hydrate (about 13%),
g-terpinene (8.5%12.17%), and -terpinene (5.05%
8.62%) (Figure 3). Mycorrhization resulted in some
changes in the percentages of essential oil components.
The EEZ 35 strain increased the content of limonene,
sabinene, and linalyl acetate, and decreased the content of

4. Discussion
AMF are integral and functional parts of plants roots,
playing a central role in the natural attenuation of metal
toxicity in their hosts. It is known that AMF colonization
promotes root growth and creates an absorptive structure
with a very high surface area of transfer of nutrients

Table 2. Correlation coefficients (r) between antioxidant capacities (DPPH and FRAP) and total
phenolic compounds and flavonoids.

DPPH I%
FRAP (mol Fe g )
2+

Total phenolic concentrations

Flavonoid concentrations

0.846*

0.582*

0.739*

0.953*

* Significant at P 0.05.
Terpinen-4-ol
cis -Sabinene hydrate
g -Terpinene
a -Terpinene
a-Terpineol
trans- Sabinene hydrate

ab

Caryophyllene E

Sabinene
p- Mentha-2,4(8) - diene
Limonene
Unknown diterpene a

c
b cd
a
a
b

Sabinene hydrate acetate-trans

0

aab
ab
b

Linalyl acetate

Myrcene

a
a c
ab
c
d

a
a b
c
ac
b
ac
ba

bb

a b
b
c

a aa
a

a
a
aa
c

NM
EEZ - 35
EEZ - 54

c
b
b
c

EEZ - 55

10

15

20

25

30 %

Content of main compounds in Origanum majorana L. essential oil (%)

Figure 3. The main compounds of essential oil (%) grown on heavy metal-polluted soil;
Values are means SE, n = 6; letters in common within a graph indicate no significant
differences assessed by Fisher LSD test (P 0.05) following ANOVA.

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HRISTOZKOVA et al. / Turk J Biol

between plant and fungus. Many species belonging to

the Lamiaceae form arbuscular mycorrhizal associations.
Positive effects of mycorrhization on the growth of
essential-oil-containing plants were reported with Ocimum
basilicum L. (Khaosaad et al., 2006) and Salvia officinalis L.
(Geneva et al., 2010). The first data showing that oregano
is highly mycorrhizal (more than 50%) were reported
by Khaosaad et al. (2006). The authors investigated the
effects of root colonization by Glomus mosseae on the yield
and quality of essential oils in three oregano genotypes
(Origanum vulgare L.). Our results indicate that the AMF
strains, depending on the genotype, have varying ability to
establish an effective mycorrhizal association with the host
plant and have an influence over the plant metabolism.
As Zhang et al. (2010) reported, AMF increased lead
accumulation in the roots, as did the EEZ 35 mycorrhizal
strain. Thus, AM fungal inoculation could attenuate the
oxidative stress of lead to Zea mays seedlings. On the other
hand, the role of AMF in decreasing Pb concentrations in
both the roots and the shoots was shown with the EEZ 54
and EEZ 55 inoculations. These strains, however, originate
from a soil with naturally high levels of metals; they are
evolutionarily more adapted to manage higher metal
concentrations and to reduce the uptake of metals by
plants. AMF occur in the soil of most ecosystems, including
polluted soils. By acquiring phosphate, micronutrients,
and water, and delivering a proportion to their hosts,
they enhance the hosts nutritional status. Similarly, heavy
metals are taken up via the fungal hyphae, where they can
be immobilized or transported to the plant. Thus, in some
cases mycorrhizal plants experience enhanced heavy metal
uptake and root-to-shoot transport, while in other cases
AMF contribute to heavy metal immobilization within
the hyphal walls and in the soil. The effects of mycorrhizal
colonization on remediation of contaminated soils depend
on the plantfungusheavy metal combination and are
influenced by soil chemical and physical conditions
(Siddiqui et al., 2008). According to Azcon et al. (2010),
AMF symbiosis may contribute to phytoremediation via
strategies such as HM sequestration or accumulation,
keeping metal concentrations in the plants below critical
values and improving plant growth and nutrition. The
specific role of AMF in the host exposure to metal stress
and in the progression of the host stress response depends
on a variety of factors, including plant species and
ecotype, fungal species and ecotype, and metal availability
(Pawlowska et al., 2004).
Antioxidant capacity is used as a parameter for
nutritional plants characterization and their bioactive
components (Ayari et al., 2013). Some authors (Schlesier
et al., 2002) strongly recommend using at least two
methods to assess the antioxidant activity of plant extracts
due to differences between the investigated test systems.
Correlation coefficient tests between antioxidant activity

466

(DPPH and FRAP) and total phenols and flavonoids in the

above-ground plant parts of O. majorana were performed.
Higher correlation coefficients for FRAP were obtained in
comparison with the DPPH test system. Oxidative stress
due to heavy metal toxicity resulted in an increase in the
activities of antioxidant metabolites such as phenolic
compounds that participate in quenching reactive oxygen
species (Kovaik et al., 2009). Our results indicate that
the O. majorana antioxidant potential, defined by the
concentration of phenolic compounds and flavonoids,
considerably increases as a result of EEZ 35 and EEZ
54 application. These results confirm previous reports
showing that total phenols and flavonoids contribute
significantly to the antioxidant capacity of sweet marjoram
(Vagi et al., 2005; Sellami et al., 2009; Ayari et al., 2013).
Besides being radical scavengers, flavonoids are also able
to function as chelators for metals (Brown et al., 1998).
Khaosaad et al. (2006) reported that AMF increased
essential oil concentrations in two of three tested oregano
genotypes. Similar results were obtained by Copetta et al.
(2006) in studies with Ocimum basilicum L. Our results are
in an agreement with the reports by Vagi et al. (2005) that
the most abundant constituent of O. majorana essential oil
is terpene-4-ol, irrespective of growth conditions. Hussain
et al. (2011) pointed out that the major constituents of
O. majorana L. grown in Pakistan were in the following
order: terpene4ol > linalool > linalyl-acetate > limonene
> -terpineol. Our results showed a different order.
The most important finding of the present research is
that AMF can keep metal concentrations in aromatic plants
at low levels and alleviate harmful effects caused by heavy
metal pollution. With respect to food quality, the cultivation
of O. majorana L. plant products minimally treated
with synthetic fertilizers requires a suitable and efficient
AMF strain. The mycorrhizal status is best manifested
in the plant roots inoculated with EEZ 54, followed by
the mycorrhization with EEZ 55. The inoculation with
the AMF strains of EEZ 54 and EEZ 55, isolated from a
place with naturally high levels of metals, reduced the Pb
concentration in marjoram shoots and roots. The highest
value of total identified essential oils was observed in EEZ
35 plants, where Pb concentration in plant tissues was not
diminished in comparison with the nonmycorrhizal plants.
Therefore, the strains isolated from a polluted habitat are
not adapted to block the uptake of heavy metals by plants.
The O. majorana antioxidant potential is mainly defined
by its high concentrations of phenolic compounds and
flavonoids. Concentration alterations of two of the five most
abundant essential oil compounds were observed as a result
of mycorrhizal colonization.
The beneficial effects of mycorrhizal fungi on
biologically active compounds, including essential oil
composition, in aerial plant parts are important to support
the development of organic and nonpollution agriculture.

HRISTOZKOVA et al. / Turk J Biol

Acknowledgments
This
work
was
supported
by
grant
BG051PO001-3.3.06-0025, financed by the European
Social Fund and Operational Programme, Human
Resources Development (20072013), and co-financed

by the Bulgarian Ministry of Education and Science.

Arbuscular mycorrhizal fungi strains were kindly provided
by Prof Concepcin Azcn Gonzlez de Aguilar and Prof
Rosario Azcn from Estacin Experimental del Zaidn,
CSIC, Spain.

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